The goal of this project is to develop a 384-well cell migration assay suitable for high throughput screening (HTS) of chemical libraries for cancer therapeutics. Current cell migration assays are not feasible in HTS settings;they are not robust, reproducible or cost-effective to perform. Fundamental changes to Platypus Technologies'OrisTM 96-wellcell migration assays, as described in this proposal, will make the proposed HTS 384-well assay compatible with automated liquid handling systems and high content screening (HCS) platforms. These enablements will dramatically improve testing throughput (allowing increased numbers of compounds to be tested) and efficiency (reducing hands- on time) while markedly reducing the cost per assay. The availability of a 384-well HTS cell migration assay that requires minimal numbers of cells, minute volumes of test compounds and reduced operator time will transform drug development research for cancer therapeutics. The proposed assay, offering transformative improvements over existing technologies, will be amenable to primary screens for cell migration inhibitors and will also permit subsequent secondary screens in the same assay wells for multiplexed probing of inhibitor effects on target molecules, viability and morphology using high content screening instruments. This HTS assay is based on the innovative use of a temporary cell exclusion zone comprised of a non-toxic, biocompatible polymer that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are seeded and attach at the perimeter of the well, and the polymeric exclusion zone dissolves to reveal a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay currently provides 96 wells, populated with silicone stoppers to create exclusion zones, for investigating the effects of cell movement modulators. Using the Oris" assay, we have demonstrated measurable migration of A-549 cells and reported z-factors of >0.46 to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We have further shown the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a dissolvable polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the current, stopper-based Oris" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this 2-year project, we propose to develop a 384-well cell migration assay that allows for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will be validated in collaboration with Dr Andreas Vogt at the University of Pittsburgh's Drug Discovery Institute, a HTS facility. The assay will enjoy a wide range of compatibility with a variety of HCS platforms for quick data retrieval. Our intended product will dramatically streamline the drug discovery process to facilitate rapid screening of molecular libraries for development of therapeutics that block cancer cell metastasis.